Anticancer compositions, and methods of making and using the same

ABSTRACT

Compositions, containing a ruthenium(III) complex and a heterocycle, methods for their manufacture, medicament containing these compositions and a kit. The invention relates to a composition (A), obtained by reacting a compound of the general formula (I) 
 
M 3−n−p−2pr [RUX 6−n−p−q−2r B n (H 2 O) p (OH) q (O) r ] 2r+1   (I) 
with a compound of the general formula (II) 
 
B′(HX′) s   (II). 
Furthermore, the invention relates to a composition (B), obtained by mixing a compound of the general formula (III) 
 
(B′H) 3−n−p−2pr [RUX 6−n−p−q−2r B n (H 2 O) p (OH) q (O) r ] 2r+1   (III) 
with a compound of the general formula (IV) 
 
MX′  (IV).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/627,519filed on Jul. 25, 2003, which is a continuation of InternationalApplication No. PCT/EP02/00863, filed Jan. 28, 2002, the content of bothof which is incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

This invention relates to compositions which include a ruthenium(III)complex and a heterocycle and also to their application as medicamentsfor the treatment of cancer illnesses.

The possible applicability of ruthenium complexes in the treatment ofcancer illnesses has been discussed by M. J. Clarke, ACS Symp. Ser. 140(1980) 157-180.

Complex compounds of trivalent ruthenium with pyrazol and imidazol havebeen described by F. Kralik et al., Collection Czechoslov. Chem. Commun.26 (1961) 1298 and by B. K. Keppler et al., Inorg. Chem., 26 (1987)4366-4370.

Furthermore, ruthenium(III) complexes with indazol and dimethylsulfoxide have been investigated by B. K. Keppler et al., AnticancerRes., 9 (1989) 761-766, G. Mestroni et al., J. Am. Chem. Soc. 111 (1989)7068-7071 and G. Mestroni et al., Inorg. Chem., 34 (1995) 4722-4734.

The tumor-inhibiting properties of ruthenium(III) complexes with a basicheterocycle, such as indazol, are also described in Eur. J. Inorg. Chem.1999, 9, pp. 1551-1555, Met.-Based Drugs 1994, 1 (2-3), pp. 145-150 andJ. Cancer Res. Clin. Oncol. 1992, 118(3), pp. 195-2000.

In the U.S. Pat. No. 4,843,069 medicaments are described which containruthenium(III) complexes with a monocyclic or multi-cyclic basicheterocycle. The complexes are suitable for cancer therapy, but aredifficult to dissolve in water and are therefore not lyophilisable.

To avoid this disadvantage, WO97/36595 discloses ruthenium(III)complexes with an alkali metal cation or with ammonia which are easilysoluble in water and which are also effective in the treatment of cancerillnesses. However, these compounds have the disadvantage that theyexhibit a lower effectiveness than ruthenium complexes according to U.S.Pat. No. 4,843,069.

BRIEF SUMMARY OF THE INVENTION

Therefore, the object of the invention is to avoid the disadvantagesmentioned above and to provide a composition which is easilywater-soluble and which exhibits a high effectiveness in the treatmentof cancer illnesses.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofpreferred embodiments of the invention, will be better understood whenread in conjunction with the appended drawings. For the purpose ofillustrating the invention, there is shown in the drawings embodimentswhich are presently preferred. It should be understood, however, thatthe invention is not limited to the precise arrangements andinstrumentalities shown.

In the drawings:

FIG. 1 shows a comparison of the cytotoxic effects of KP 1019, of thecorresponding sodium salt KP 1339, indazol as well as the equimolarmixture of KP 1339 and indazol (administered as a solution in aconventional cell culture medium MEM, supplemented with 10% of fetalbovine serum, without the addition of organic solvents or dissolvingagents) on the human line of tumor cells SW480 (colon carcinoma) in theMTT assay with continuous 96 hour exposure to the active substance.

FIG. 2 shows a comparison of the cytotoxic effects of KP 1019, of thecorresponding sodium salt KP 1339, indazol as well as the equimolarmixture of KP 1339 and indazol (administered as a solution in aconventional cell culture medium MEM, supplemented with 10% of fetalbovine serum, without the addition of organic solvents or dissolvingagents) on the human line of tumor cells CH1 (ovarian carcinoma) in theMTT assay with continuous 96 hour exposure to the active substance.

FIG. 3 shows a comparison of the cytotoxic effects of KP 1019, indazol,and of mixtures of KP 1339 and indazol in the molar ratios of 1: 1 and1:5 (administered as a solution in a conventional cell culture mediumMEM, supplemented with 10% of fetal bovine serum, without the additionof organic solvents or dissolving agents) on the human line of tumorcells SW480 (colon carcinoma) in the MTT assay with continuous 96 hourexposure to the active substance.

FIG. 4 shows a comparison of the cytotoxic effects of KP 1019, indazol,and of mixtures of KP 1339 and indazol in the molar ratios of 1: 1 and1:5 (administered as a solution in a conventional cell culture mediumMEM, supplemented with 10% of fetal bovine serum, without the additionof organic solvents or dissolving agents) on the human line of tumorcells CH1 (ovarian carcinoma) in the MTT assay with continuous 96 hourexposure to the active substance.

The manufacture of the compounds of the general formula (I) can takeplace according to the instructions described in WO 97/36595.Furthermore, the manufacture of the compounds of the general formula(III) can take place as shown in J. Cancer Res. Clin. Oncol. 1992,118(3), pp. 195-200. The manufacture of the compounds of the formulae(II) and (IV) takes place according to known methods.

DETAILED DESCRIPTION OF THE INVENTION

This object is solved by a composition (A), obtained by reacting acomplex compound of the general formula (I)M_(3−n−p−2pr)[RUX_(6−n−p−q−2r)B_(n)(H₂O)_(p)(OH)_(q)(O)_(r)]_(2r+1)  (I)where

-   -   M is an alkali metal cation or ammonia,    -   B is a monocyclic or multi-cyclic basic heterocycle with one or        more nitrogen atoms,    -   X is a halide, pseudo-halide, HCO₃ ⁻, or RCOO⁻, in which R is a        substituted or unsubstituted C₁-C₆-alkyl or C₂-C₆-alkenyl or a        substituted or unsubstituted aryl, n=1 or 2,    -   p, q=0 or 1 or (if r=0.5) 0 or 0.5, and    -   r=0 or 0.5,

with a compound of the formula (II)B′(HX′),  (II)

where

-   -   B′ is a monocyclic or multi-cyclic basic heterocycle with one or        more nitrogen atoms,    -   X′ is a halide, pseudo-halide, HCO₃ ⁻, or RCOO⁻, in which R is        hydrogen or a substituted or unsubstituted C₁-C₆-alkyl or        C₂-C₆-alkenyl or a substituted or unsubstituted aryl, phosphate,        sulphate and/or acetate, and    -   s is an integer of 1 or more.

The object is also solved by a composition (B), obtained by mixing acomplex compound of the general formula (III)(B′H)_(3−n−p−2pr)[RUX_(6−n−p−q−2r)B_(n)(H₂O)_(p)(OH)_(q)(O)_(r)]_(2r+1)  (III)

where each B, B′ is a monocyclic or multi-cyclic basic heterocycle withone or more nitrogen atoms,

-   -   X is a halide, pseudo-halide, HCO₃ ⁻, or RCOO⁻, in which R is a        substituted or unsubstituted C₁-C₆-alkyl or C₂-C₆-alkenyl or a        substituted or unsubstituted aryl,    -   n=1 or2,    -   p, q=0 or 1 or (if r=0.5) 0 or 0.5, and    -   r=0 or 0.5,

with a compound of the formula (IV)MX′  (IV)

where

-   -   M is an alkali metal cation or ammonia and    -   X′ is a halide, pseudo-halide, HCO₃ ⁻, or RCOO⁻, in which R is        hydrogen or a substituted or unsubstituted C₁-C₆-alkyl or        C₂-C₆-alkenyl or a substituted or unsubstituted aryl, phosphate,        sulphate or acetate.

B and/or B′ in the formulae (I), (II) or (III) can be purine, adenine,guanine, cytosine, indazol, imidazol, pyrazol, pyridine, pyrimidine,pyridazine, pyrrol, tetrazol and/or triazine which can be substituted byone or more substituents, selected from the group consisting of:hydroxyl, amino, halogen, C₁-C₄-alkyl, C₂-C₄-alkenyl, C₁-C₄-alkylmercapto, formyl-, C₁-C₄-alkoxy carbonyl, C₁-C₄-alkoxycarbonyl-C₁-C₄-alkylene, di-C₁-C₄-alkyl-amino,di-C₁-C₄-alkyl-amino-C₁-C₄-alkylene, di-C₁-C₄-alkyl-amino-carbonyl,di-C₁-C₄-alkyl-amino-carbonyl-C₁-C₄-alkylen, hydroxy imino methine,phenyl, benzyl, benzoyl, pyrrolidino, piperidino, pyrrol-1-yl andpyrrol-1-yl-C₁-C₄-alkylene,

or a ring

where

-   -   R^(1′) is hydrogen, sodium, C₁-C₄-alkyl or phenyl and    -   R^(2′) is hydrogen, C₁-C₄-alkyl, amino or phenyl, or R^(1′) and        R²⁺ together represent a group —(CH₂)_(s)— where s is an integer        from 4 to 8,    -   W is nitrogen or CR^(3′), where R^(3′) is a hydrogen,        C₁-C₄-alkyl, amino or phenyl, preferably hydrogen or methyl,    -   Y is nitrogen or CR^(4′) where R^(4′) is a hydrogen,        C₁-C₄-alkyl, amino or phenyl, preferably hydrogen or methyl, and    -   Z is nitrogen or CR^(5′) where R^(5′) is a hydrogen,        C₁-C₄-alkyl, amino or phenyl, preferably hydrogen or methyl,        whereby the ring is bonded to the basic heterocycle B or B′ by        at least one of the substituents R¹ and R².

Preferably at least one of the groups R^(3′), R^(4′) or R^(5′) is ahydrogen.

Preferably the at least one substituent is selected from the groupconsisting of chlorine, diethylamino, dimethylamino andpyrrol-1-yl-methyl.

In a preferred embodiment B and/or B′ in the formulae (I), (II) or (III)can be 1-methylimidazol, 4-methylimidazol, 4-methylpyrazol, 1-sodiumpyrazol, 1-phenyltetrazol or 5-phenyltetrazol which can be substituted,preferably in position 4 by one or more substituents, as defined above.

In a further preferred embodiment B and/or B′ in the formulae (I), (II)or (III) are imidazol, pyrazol, triazol or indazol, with imidazol,triazol or indazol being particularly preferred, but more preferablytriazol or indazol, especially indazol.

B′ and B can be the same, i.e. they can represent the same heterocycle.

Furthermore, M in the formulae (I) and (IV) is preferably lithium,sodium or potassium, especially sodium.

In the preferred embodiments X and/or X′ in the formulae (I), (II),(III) or (IV) are chlorine or bromine, especially chlorine. In anotherpreferred embodiment X′ in the composition (A) or (B) corresponds to X.

The molar ratio of the compound of formula (I) to the compound offormula (II) in the composition (A) according to the invention ispreferably <1, a ratio between 1:1.1 and 1:10 is particularly preferred,and especially 1:2 and 1:5.

The molar ratio of the compound of formula (III) to the compound offormula (IV) in the composition (B) according to the invention ispreferably a ratio between 1:2 and 1:30, with 1:5 and 1:15 beingparticularly preferred, with 1:10 being especially preferred.

In a preferred embodiment the compound of formula (I) is sodiumtrans-tetrachlorobis (1 H-indazol)-ruthenate(III).

The compound of formula (II) is preferably indazolium hydrochloride.

The compound of formula (III) is preferably indazoliumtrans-[tetrachlorobis-(indazol)ruthenate(III)].

The compound of formula (IV) is preferably sodium chloride.

The composition (A) or (B) according to the invention can be present inthe form of an aqueous solution.

The composition (B) can be obtained by attrition of a compound of theformula (III) with a compound of formula (IV) in mortars or mills, suchas air stream or ball mills.

It was surprisingly found that the availability of a compound of thegeneral formula (III) in aqueous solution can be improved by mixing witha compound of the general formula (IV) while retaining the composition(B).

It was also surprisingly found that a compound of the general formula(I), which is slightly soluble in water and lyophilisable, offerssuitable formulation methods by reaction with a compound of the generalformula (II) to form a composition (A) according to the inventionsuitable for the treatment of cancer illnesses (see Examples 1-3 andFIGS. 1-4).

Furthermore, the object of the invention is solved by a medicament whichcontains the compositions (A) and/or (B) according to the invention.

The compositions (A) and/or (B) can furthermore be employed in themanufacture of a medicament for the prophylaxis and/or treatment ofcancer illnesses.

The medicament according to the invention is described in more detail asfollows.

The medicaments according to the invention are primarily administeredintravenously, but also intramuscularly, intraperitoneally,subcutaneously or perorally. External application is also possible.Administration through intravenous injection or infusion is preferred.

The pharmaceutical preparations are produced by known methods wherebythe composition according to the invention is used on its own or, wherenecessary in combination with suitable pharmaceutical carriersubstances. If the pharmaceutical preparations contain pharmaceuticalcarrier substances as well as the active substance, the content ofactive substance in these mixtures is 0.1 to 99.5%, preferably 0.5 to95% by weight of the total mixture.

The active substance can be applied in any suitable formulation with therequirement that the formation or maintenance of a sufficient level ofactive substance is ensured. This can, for example, be achieved throughoral or parenteral administration in suitable doses. Advantageously,there is the pharmaceutical preparation of the active substance in theform of standard doses, which are adapted to the desired administration.A standard dose may be, for example, a tablet, dragee, capsule,suppository or a measured volume of a powder, a granulate, solution,emulsion or suspension.

A “standard dose” in the sense of this invention is taken to mean aphysically determined unit which contains an individual quantity of theactive constituent in combination with a pharmaceutical carriersubstance and its content of active substance corresponds to a fractionor multiple of a therapeutic single dose. A single dose preferablycontains the quantity of active substance which is administered duringan application and which normally corresponds to a whole, half, third orquarter of the daily dose. If only a fraction, such as half or quarterof the standard dose is needed for a single therapeutically administereddose, then the standard dose is advantageously divisible, e.g. in theform of a tablet with a dividing groove.

The medicaments according to the invention can, if they are available instandard doses and intended for application, e.g. on persons, containabout 0.1 to 500 mg, preferably 10 to 200 mg and particularly 50 to 150mg of active substance.

Generally in human medicine, the active substance(s) are administered ina daily dose of 0.1 to 5, preferably 1 to 3 mg/kg of body weight, wherenecessary in the form of a number, preferably 1 to 3, of single intakesfor achieving the desired results. A single intake contains the activesubstance(s) in quantities of 0.1 to 5, preferably 1 to 3 mg/kg of bodyweight. With oral treatment similar dosages can be applied.

The therapeutic administration of pharmaceutical preparations can occur1 to 4 times daily at specified or varying time points, e.g. in eachcase before meals and/or in the evening. However, it may be necessary todeviate from the quoted dosages depending on the type, body weight andage of the individual to be treated, the type and severity of theillness, the type of preparation and the application of thepharmaceutical preparations as well as the time period or intervalwithin which the administration occurs. Consequently, in some cases itmay be sufficient to use less than the amount of active substancementioned above, whereas in other cases the above listed quantities ofactive substance must be exceeded. It may also be practicable toadminister the pharmaceutical preparations only once or at intervals ofseveral days.

The specification of the necessary optimum dosage and type ofapplication of the active substance can be made by any specialist basedon his specialist knowledge.

The pharmaceutical preparations normally comprise the compositionaccording to the invention and non-toxic, pharmaceutically compatiblemedication carriers which are employed as admixtures or diluting agentsfor example in solid, semi-solid or liquid form or as a means ofenclosure, for example in the form of a capsule, a tablet coating, a bagor another container for the therapeutically active constituent. Acarrier material may, for example, act as an agent for the ingestion ofthe medicament by the body, as a formulation agent, sweetener, tastemodifier, colorant or as preservative.

For oral application, for example, tablets, dragees, hard and softcapsules, for example of gelatine, dispersible powder, granulate,aqueous and oily suspensions, emulsions, solutions and syrups can beemployed.

Tablets can contain inert binding agents, e.g. calcium carbonate,calcium phosphate, sodium phosphate or lactose; granulation anddistributing agents, e.g. maize starch or alginates; binding agents,e.g. starch, gelatine or arabine; and lubricating agents, e.g. aluminiumor magnesium stearate, talcum or silicone oil. They can also be providedwith a coating which is produced such that it causes delayed release andresorption of the pharmaceutical preparation in the gastro-intestinaltract, so that, for example, improved compatibility, assimilation orretardation is achieved. Gelatine capsules may contain thepharmaceutical substance with a solid, e.g. calcium carbonate or kaolinor an oily, e.g. olive, peanut or paraffin oil dilution agent.

Aqueous suspensions can contain suspension agents, e.g. sodiumcarboxymethyl cellulose, methyl cellulose, hydroxypropyl cellulose,sodium alginate, polyvinyl pyrrolidon, traganth rubber or arabine;dispersant or wetting agents, e.g. polyoxyethylene stearate,heptadeca-ethylene-oxycatanol, polyoxyethylene sorbitol-monooleate, orlecithin; preservatives, e.g. methyl- or propylhydroxy-benzoate; tastemodifiers; sweeteners, e.g. saccharose, lactose, sodium cyclamate,dextrose, invert sugar syrup.

Oily suspensions may be, for example, peanut, olive, sesame, coconut orparaffin oil and thickening agents, such as beeswax, high melting pointwax or cetyl alcohol; also sweeteners, taste modifiers and antioxidants.

Powder and granulates dispersible in water may contain the compositionaccording to the invention in a mixture with dispersing, wetting andsuspension agents, e.g. those mentioned above as well as withsweeteners, taste modifiers and colorants.

Emulsions can, for example, contain olive, peanut or paraffin oil aswell as emulsifying agents such as arabine, traganth rubber,phosphatides, sorbitan monooleate, polyoxyethylene sorbitan monooleateand sweeteners and taste modifiers.

Aqueous solutions can contain preservatives, e.g. methyl- orpropylhydroxybenzoates; thickening agents; taste modifiers; sweeteners,e.g. saccharose, lactose, sodium cyclamate, dextrose, invert sugar syrupas well as taste modifiers and colorants.

For the parenteral application of pharmaceutical substances sterileinjectable aqueous solutions, isotonic salt solutions or other solutionscan be used.

As a menstruum for infusion any suitable solution can be used. Water andHaemaccel® are preferred.

The method according to the invention for the production of thecomposition (A) includes the reaction of a complex compound of theformula (I) with a compound of the formula (II). The reaction preferablytakes place in an aqueous solution.

The method according to the invention for the production of thecomposition (B) includes mixing a complex compound of the formula (III)with a compound of the formula (IV). Here, a compound of the formula(III) is put through attrition with a compound of the formula (IV), e.g.in the mortar or in ball mills.

Furthermore, a kit (A) is made available which comprises a receptaclewith a compound of the formula (I)M_(3−n−p−2pr)[RuX_(6−n−p−q−2r)B_(n)(H2O)_(p)(OH)_(q)(O)_(r)]_(2r+1)  (I)

where

-   -   M is an alkali metal cation or ammonia,    -   B is a monocyclic or multi-cyclic basic heterocycle with one or        more nitrogen atoms,    -   X is a halide, pseudo-halide, HCO₃ ⁻, or RCOO⁻, in which R is a        substituted or unsubstituted C₁-C₆-alkyl or C₂-C₆-alkenyl or a        substituted or unsubstituted aryl,    -   n=1 or 2,    -   p, q=0 or 1 or (if r=0.5) 0 or 0.5, and    -   r=0 or 0.5,

as well as a receptacle with a compound of the formula (II)B′(HX′)_(s)  (II)

where

-   -   B′ is a monocyclic or multi-cyclic basic heterocycle with one or        more nitrogen atoms,    -   X′ is a halide, pseudo-halide, HCO₃ ⁻, or RCOO⁻, in which R is        hydrogen or a substituted or unsubstituted C₁-C₆-alkyl or        C₂-C₆-alkenyl or a substituted or unsubstituted aryl, phosphate,        sulphate, acetate, and    -   s is an integer of 1 or more.

In a preferred embodiment X′ in kit (A) is the same radical as X and Bthe same radical as B′.

Also, a kit (B) is made available which comprises a receptacle with acompound of the formula (III)(B′H)_(3−n−p−2pr)[RUX_(6−n−p−q−2r)B_(n)(H₂O)_(p)(OH)_(q)(O)_(r)]_(2r+1)  (III)

where

-   -   B, B′ is a monocyclic or multi-cyclic basic heterocycle with one        or more nitrogen atoms,    -   X is a halide, pseudo-halide, HCO₃ ⁻, or RCOO⁻, in which R is a        substituted or unsubstituted C₁-C₆-alkyl or C₂-C₆-alkenyl or a        substituted or unsubstituted aryl,    -   n=1 or 2,    -   p, q=0 or 1 or (if r=0.5) 0 or 0.5, and    -   r=0 or 0.5,

as well as a receptacle with a compound of the formula (IV)MX′  (IV)

where

-   -   M is an alkali metal cation or ammonia,    -   X′ is a halide, pseudo-halide, HCO₃ ⁻, or RCOO⁻, in which R is        hydrogen or a substituted or unsubstituted C₁-C₆-alkyl or        C₂-C₆-alkenyl or a substituted or unsubstituted aryl, phosphate,        sulfate or acetate.

To the radicals X, X′, B and B′ the same applies as for the above kit(A).

In the following the kit (A) according to the invention is explained inmore detail.

In the clinic the application of the composition (A) according to theinvention on the patient can take place through the provision of a kitwhich contains an ampoule with a compound of the formula (I), preferablyas sodium salt, as well as separately an infusion solution whichcontains an equimolar or also a higher concentration of a compound ofthe formula (II), preferably as hydrochloride. Before the application onthe patient, the content of the ampoule dissolved in water can beinjected into the infusion bottle. In this way the composition (A)according to the invention is formed which can then be immediately usedon the patient.

In the following the kit (B) according to the invention is explained inmore detail.

In the clinic the application of the composition (B) according to theinvention on the patient can take place through the provision of a kitwhich contains an ampoule (1) with a compound of the formula (III),preferably as indazolium salt, as well as separately an ampoule (2) witha compound of the formula (IV), e.g. sodium chloride, as well as amenstruum for the infusion. Before the application on a patient thecontent of the ampoule (1) is put through attrition, e.g. in a mortarwith the content of ampoule (2) under retention of the composition (B)according to the invention. Then the compound (B), dissolved in themenstruum for infusion, can be injected into the infusion bottle andimmediately used on the patient.

In the following the invention is explained based on some examples.

EXAMPLE 1

The compound sodium trans-[RuCl₄(ind)₂], KP1339, which is easilywater-soluble and lyophilisable was reacted with indazoliumhydrochloride to form a composition (A) according to the invention,containing indazolium trans-[tetrachlorobis(1H-indazol)-ruthenate(III)](KP 1019) and sodium chloride (see FIG. 1).

FIG. 1: Formation of a composition (A) according to the invention,containing indazolium trans-[tetrachlorobis(1H-indazol)-ruthenate(III)](KP 1019) and sodium chloride, by the reaction of KP 1339 and indazoliumhydrochloride.

EXAMPLE 2

Cytotoxicity investigations were carried out on the composition obtainedaccording to Example 1. The composition was obtained by reacting thesodium salt KP1339, which exhibits a water-solubility of more thanthirty times higher than KP 1019, with indazolium hydrochloride in theinfusion solution immediately before administration.

Since experiments on the stem cells of human solid tumors which werebred on as nude mice xenografts, and on continuous human tumor celllines corresponded in showing that KP 1339 in vitro exhibits clearlyweaker antiproliferative effectiveness than KP 1019, clarification wasobtained in further experiments of how far the stronger activity of KP1019 can be restored by the addition of indazol to solutions of KP 1339.

The tumor inhibiting activity of the equimolar mixture of KP 1339 andindazol proved to be identical to that of the original KP 1019 (seeFIGS. 1 and 2), whereas pure KP 1339 was three to four times lesseffective than KP 1019. This has been confirmed on two tumor cell lines(SW480, CH1) and reproduced four times. In these experiments the lowereffectiveness of KP 1339 in comparison to KP 1019 was confirmed.

EXAMPLE 3

In the first experiments with compositions which had been obtained byreacting KP 1339 with indazolium hydrochloride in the molar ratio of1:1, it could be shown as a result that the cytotoxic activity of theoriginal KP 1019 was attained. Further experiments gave insight into themanner in which an excess of indazol affected the cytotoxicity.

The cytotoxic effects of KP 1019 and combinations of KP 1339 and indazolin different molar ratios (administered as solution in the conventionalcell culture medium MEM, supplemented with 10% fetal bovine serum,without the addition of organic solvents or dissolving agents) have beencomparatively investigated on the human line of tumor cells SW480 (coloncarcinoma) and CH1 (ovarian carcinoma) in the MTT assay under theapplication of a continuous 96 hour exposure to the active substance.

It has been surprisingly shown that the tumor inhibiting activity can befurther increased by the addition of an excess of indazol. A mixture ofKP 1339 and indazol in the molar ratio of 1:5 gave a two to five timesincrease in the cytotoxicity compared to KP 1019 (see FIGS. 3 and 4). Amolar ratio of 1:2 is already sufficient to exceed the effectiveness ofthe original KP 1019. Indazol itself only exhibits a cytotoxic effect inhigher concentrations (>1 mM). The increase in the tumor inhibitingactivity of the solutions which contain an excess of indazol cannottherefore be explained by a purely additive effect of the components,but is rather attributable either to a synergistic effect, a positiveeffect of the free indazol on the stability of the complex or on anunknown mechanism. The discontinuous trace of the dosage/effect curve onthe cell line SW480 which proved to be strictly reproducible in fiveindependent experimental runs indicates a complex interaction between KP1339 and indazol.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications within the spirit and scope of thepresent invention as defined by the appended claims.

1. A process of providing a composition comprising a mixture of: (i) acompound of the general formula (III):(B′H)_(3−n−p−2pr)[RuX_(6−n−p−q−2r)B_(n)(H₂O)_(p)(OH)_(q)(O)_(r)]_(2r+1)  (III)wherein B and B′ are independently a monocyclic or multi-cyclic basicheterocycle with one or more nitrogen atoms, X represents a halide,pseudo-halide, HCO₃ ⁻, or RCOO⁻ in which R represents a substituted orunsubstituted C₁-C₆-alkyl or C₂-C₆-alkenyl or a substituted orunsubstituted aryl, n=1 or 2, r=0, and each of p and q=0 or 1, or r=0.5and each of p and q=0 or 0.5; and (ii) a compound of the formula (VI):MX′  (IV) wherein M represents an alkali metal cation or ammonia, and X′represents a halide, pseudo-halide, HCO₃ ⁻, or RCOO⁻ in which R ishydrogen or a substituted or unsubstituted C₁-C₆-alkyl or C₂-C₆-alkenylor a substituted or unsubstituted aryl, phosphate, sulphate or acetate,said process comprising: reacting a compound of the formula (I)M_(3−n−p−2pr)[RuX_(6−n−p−q−2r)B_(n)(H₂O)_(p)(OH)_(q)(O)_(r)]_(2r+1)  (I)with a compound of the formula (II)B′(HX′)_(s)  (II) wherein M, B, X, B′, X′, n, p, q, r, and s are asdefined above.
 2. The process according to claim 1, wherein the molarratio of the compound of the formula (I) to the compound of the formula(II) is less than
 1. 3. The process according to claim 1, wherein themolar ratio of the compound of the formula (I) to the compound of theformula (II) is from 1:1.1 to 1:10.
 4. The process according to claim 1,wherein the molar ratio of the compound of the formula (I) to thecompound of the formula (II) is from 1:2 to 1:5.